DESCRIPTION (applicant's abstract): T cell receptor (TCR) cross-reactivity towards diverse ligands is an inherent property of cellular immunity, which enables the opposing T cell functions of self-tolerance versus reactivity towards numerous foreign antigens. TCR degeneracy occurs at the levels of 1) peptide-MHC (pMHC) recognition, where a single TCR can recognize a range of highly related, or minimally homologous peptide ligands, and 2) T cell activation, where a TCR can fine-tune its stimulatory response to agonist, partial agonist and antagonist peptides. A deleterious practical manifestation of degeneracy occurs in autoimmune diseases like multiple sclerosis (MS), where autoreactive T cells productively interact with class II MHC molecules presenting processed self-peptides, such as myelin basic protein (MBP). The aberrant autoreactivity of these T cells is likely a result of their 43 TCRs recognizing shared structural features between normally tolerated self-peptides and antigenic foreign pathogens, in a phenomenon termed "molecular mimicry." Underscoring this hypothesis is the observation that, in both human MS and the experimental allergic encephalomyelitis (EAE) murine model of MS, microbial peptides have been identified that activate MBP-reactive, pathogenic T cells and in the case of EAE, induce the disease. There have been no biophysical studies on autoimmune TCR cross-reactivity with defined self and microbial antigens. We propose to apply complementary biochemical, biophysical and functional approaches to understanding degenerate T cell recognition in the well-defined EAE system. We will define the molecular interactions in the TCR/pMHC interface for a series of cross-reactive complexes using soluble, recombinant TCR and pMHC molecules that will be utilized for biochemical and x-ray crystallographic analysis. We will identify additional, novel peptide ligands that cross-react with the EAE TCRs by screening combinatorial libraries against these autoreactive T cell clones. Lastly, we will examine whether cross-reactive peptides differentially cluster the TCR signaling complex in the T cell/APC interface using real-time, 3-dimensional, fluorescence imaging techniques with live cells. The overall goal of these studies is to bridge the gap between the static information gained from in vitro studies of the molecular details of TCR/pMHC recognition using soluble molecules, and the dynamic activation events, which occur in vivo, on the cell-surface as a result of this complex formation.